Biomedical subjects

H G Stunnenberg

Publications and source records attributed to H G Stunnenberg.

At least 55 records · Page 3Linked to original sources

PubMed1992

Cooperativity in transactivation between retinoic acid receptor and TFIID requires an activity analogous to E1A.

In embryonal carcinoma (EC) cells retinoic acid (RA) strongly induces transcription from the RA receptor beta 2 (RAR beta 2) promoter through an RA response element (RARE) located in close proximity to the TATA box. Here we demonstrate that recombinant human TATA box-binding protein, hTFIID, and RAR functionally cooperate in transactivation of the RAR beta 2 promoter in EC cells in a strictly RA-dependent manner. We demonstrate that the core domain of hTFIID is sufficient to mediate RAR-dependent transcription and that Drosophila, but not yeast, TFIID can substitute for hTFIID. In COS cells ectopic expression of the E1A protein is a prerequisite for hTFIID and RAR to cooperate in transactivation. We propose a model for transcriptional regulation of the RAR beta 2 promoter in EC cells in which RAR, following activation by RA, functionally interacts with hTFIID via an E1A-like activity present in EC cells.

Adenovirus Early Proteins↗

PubMed1992

Identification of multiple SRF N-terminal phosphorylation sites affecting DNA binding properties.

Human serum response factor (SRF) bearing a histidine tag was expressed using vaccinia virus. The recombinant protein was purified and shown to be phosphorylated mainly in its N-terminal part. The corresponding phosphorylation sites were mapped by microsequencing and also appear to be phosphorylated in endogenous serum response factor. Four phosphorylation sites are located on serines within amino acids 77-85, while another phosphorylation site has been identified at Ser103. Mutations that considerably reduced or abolished phosphorylation at amino acids 77-85 caused a decrease in binding to the c-fos serum response element accompanied by markedly reduced association and dissociation rates. In contrast, replacing Ser103 by alanine decreased DNA binding activity without drastically affecting the on/off rates. The combination of abolishing phosphorylation at amino acids 77-85 and 103 displayed greatly reduced on/off rates of DNA binding, but the reduction of DNA binding activity was partially alleviated. None of these mutations affect either the ability to interact with p62TCF or stimulation of transcription in vitro. These findings imply possible roles for SRF phosphorylation in the regulation of c-fos transcription.

Amino Acid Sequence↗

PubMed1992

RXR alpha, a promiscuous partner of retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR), thyroid hormone receptor (T3R) and vitamin D3 receptor (VD3R) differ from steroid hormone receptors in that they bind and transactivate through responsive elements organized as direct rather than inverted repeats. We now show that recombinant RAR and T3R are monomers in solution and cannot form stable homodimeric complexes on their responsive elements. Stable binding of the receptors to their responsive elements requires heterodimerization with a nuclear factor. This auxiliary factor is tightly associated with RAR and T3R in the absence of DNA and co-purifies with both receptors. As demonstrated by extensive purification, the same auxiliary factor is required for stable DNA binding of RAR as for that of T3R; the factor also facilitates the formation of a stable VD3R-DNA complex. The auxiliary factor is identical to the retinoid X receptor alpha (RXR alpha) by biochemical and functional criteria. The identification of RXR alpha as a dimerization partner for the RARs, T3Rs and VD3R has important implications as to the function of these receptors and their ligands in development, homeostasis and neoplasia.

Animals↗

PubMed1992

The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation.

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.

Amino Acid Sequence↗

PubMed1991

Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.

Vaccinia virus has been used as a vector to express foreign genes for the production of functional and posttranslationally modified proteins. A procedure is described here that allows the rapid native purification of vaccinia-expressed proteins fused to an amino-terminal tag of six histidines. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In the case of the human serum response factor (SRF), a transcription factor involved in the regulation of the c-fos protooncogene, the vaccinia-expressed histidine-tagged SRF (SRF-6His) could be purified solely by this step to greater than 95% purity. SRF-6His was shown to resemble authentic SRF by functional criteria: it was transported to the nucleus, bound specifically the c-fos serum response element, interacted with the p62TCF protein to form a ternary complex, and stimulated in vitro transcription from the serum response element. Thus, the combination of vaccinia virus expression and affinity purification by Ni2+.NTA chromatography promises to be useful for the production of proteins in a functional and posttranslationally modified form.

Amino Acid Sequence↗

PubMed1991

Promoter melting by a stage-specific vaccinia virus transcription factor is independent of the presence of RNA polymerase.

Fractionation of an extract prepared from HeLa cells infected with vaccinia virus resulted in the separation of factors involved in vaccinia virus intermediate transcription. Two activities, VITF-A and VITF-B, in addition to the viral RNA polymerase are necessary and sufficient to direct intermediate transcription in vitro. VITF-B confers intermediate promoter specificity to an early-specific extract prepared from virus particles. A committed complex between VITF-B and the template can sequester VITF-A and RNA polymerase into a pre-initiation complex. VITF-B is further able to melt the promoter at the start site of transcription. Open complex formation is stimulated by ATP. In contrast to prokaryotic and eukaryotic pol III transcription, promoter melting is independent of the presence of RNA polymerase.

Adenosine Triphosphate↗

PubMed1991

Functional characterization of a natural retinoic acid responsive element.

Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are thought to bind as dimers to a T3 responsive element (T3REpal) comprised of inverted repeats of the half-site motif GGTCA. However, a RA responsive element (beta RARE) was previously identified in the promoter of the RAR beta 2 gene which contains two direct repeats of the motif GTTCA spaced by a six nucleotide gap. We now demonstrate the ability of RAR alpha, beta and gamma to bind to and transactivate through this element and that the two direct repeats comprise the beta RARE. Surprisingly, the GTTCA motifs rearranged to form a palindrome do not confer RA responsiveness to a heterologous promoter. Furthermore, no significant level of transactivation is detected by ligand-activated RAR through the Moloney murine leukaemia virus T3RE, which comprises two direct repeats of the sequence GGTCA/C spaced by a five nucleotide gap. Similarly, T3R does not induce gene expression through the beta RARE. This study establishes the preference of T3R to transactivate through direct repeats spaced by a five nucleotide gap as opposed to a six nucleotide gap. In contrast, RAR appears to be more flexible with respect to spacing requirements between repeats, although higher levels of transactivation are obtained through direct repeats spaced by a six nucleotide gap. Interestingly, although some elements mediate either RA or T3 induction, changing a single nucleotide in the MoMLV T3RE with a five nucleotide spacing creates a promiscuous RA/T3 responsive element.

Animals↗

PubMed1991

TFIID is required for in vitro transcription of the human U6 gene by RNA polymerase III.

We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase III transcription of the human U6 snRNA gene. Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays. First, TFIID-containing fractions stimulate U6 transcription in reactions containing rate-limiting amounts of HeLa nuclear extract. Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates. Third, TFIID can replace one of two heat labile fractions essential for U6 transcription. Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases.

Base Sequence↗

PubMed1991

Vaccinia virus capping enzyme is a transcription initiation factor.

It has previously been demonstrated that vaccinia virus capping enzyme is involved both in the formation of a 5' cap structure and in termination of early transcription. Here we show that capping enzyme has an additional activity which is required for transcription of intermediate genes. VITF-A and VITF-B have been defined as two activities which together with RNA polymerase are necessary and sufficient to transcribe intermediate genes in vitro. VITF-A and the viral capping enzyme are shown to copurify to near homogeneity. Direct evidence that capping enzyme is VITF-A was obtained by complementation of a reconstituted transcription system with viral capping enzyme expressed in Escherichia coli. Although capping enzyme is a cofactor in early transcription termination, intermediate transcription is not terminated in response to the early termination signal. Capping enzyme is shown to form a complex with RNA polymerase in the absence of VITF-B. This appears to be a prerequisite for the formation of a stable initiation complex.

DNA-Directed RNA Polymerases↗

PubMed1991

Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin.

We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Mutational analysis of a vaccinia virus intermediate promoter in vivo and in vitro.

The expression of the vaccinia virus intermediate I3 gene depends on trans-acting factors which are present in an active state prior to DNA replication. However, activation of transcription requires DNA replication in cis (J. C. Vos and H. G. Stunnenberg, EMBO J., 7:3487-3492, 1988). We have made deletion and linker scanner mutations of the I3 promoter to determine the sequence requirements for transcriptional activity and the dependence of DNA replication. The I3 promoter appears to consist of two elements which are essential and sufficient for accurate transcription initiation both in vivo and in vitro. An upstream and a downstream sequence element were defined ranging from -20 to -9 and +1 to +9, respectively. The upstream element appears to be highly homologous to a sequence in the intermediate I8 promoter. A 3-bp substitution in the upstream I3 promoter element resulted in a change of transcriptional specificity from intermediate to late. Finally, the mutations did not result in an activation of the intermediate promoter prior to DNA replication.

Animals↗

PubMed1988

Cis-acting sequences affecting the length of the poly(A) head of vaccinia virus late transcripts.

Mutations in the sequences flanking the conserved and essential TAAAT motif of vaccinia late gene promoters (consensus: T/A T/A TAAAT G Pu Pu) affect the level of expression. Introduction of a pyrimidine in the purine stretch downstream of the TAAAT motif reduces the level of RNA synthesis. Mature transcripts from the wild-type 11K late promoter have a non-contiguous 5' poly(A) leader of approximately 35 A-residues (referred to as a poly(A) head). We show here by RNA sequencing, primer extension and subsequent m7G cap selection of cDNA/RNA hybrids that the mutations affect the length of the poly(A) head but not the location of the junction between the poly(A) leader and sequences encoded in the genome. These results are consistent with a slippage mechanism underlying the process of 5' poly(A) addition, but are not in agreement with a splicing event.

Base Sequence↗

PubMed1988

High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system.

DNA Replication↗

PubMed1988

Derepression of a novel class of vaccinia virus genes upon DNA replication.

A novel class of vaccinia virus genes, called intermediate, is expressed immediately post-replication and prior to the onset of late gene transcription. Intermediate transcription is dependent on trans-acting factors which are present in an active state in virus-infected cells prior to the onset of DNA replication. Plasmid-borne intermediate genes transfected into vaccinia-virus infected cells are expressed prior to DNA replication, whereas the copies within the viral genome are repressed. DNA replication is essential for activation of viral intermediate transcription and de novo protein synthesis is not required post-replication. In contrast, activation of late transcription depends on DNA replication and continued de novo protein synthesis. Therefore, a subset of intermediate proteins is likely to be trans-activators of late gene transcription. Cell-free extracts differentially transcribe early, intermediate and late genes in a way similar to the temporal expression observed in vivo. A cascade model is discussed for the regulation of gene expression during the viral life-cycle.

Base Sequence↗

PubMed1988

Sequence and transcriptional analysis of the vaccinia virus HindIII I fragment.

The complete sequence of the vaccinia virus HindIII I fragment, which is composed of 6,498 base pairs, encodes six complete and two incomplete open reading frames (ORFs). Computer analysis revealed an amino acid sequence homology between ORF I 4 and the large subunit of the ribonucleotide reductase complex. The two small polypeptides derived from ORFs I 2 and I 5, with molecular weights of 8,500 and 8,700, respectively, have a very high hydrophobic amino acid sequence composition. S1 analysis revealed that ORF I 4 is expressed at early stages of infection, ORFs I 1, I 2, I 5, and I 7 are expressed in the late phase of infection, and ORF I 3 is constitutively expressed. Screening a vaccinia virus genomic library revealed a large vaccinia virus insert overlapping the HindIII I and O fragments which contains a previously undetected HindIII P fragment of approximately 300 base pairs. S1 analysis revealed an early (O1) and a late (O2) start site of transcription initiation located within the HindIII O fragment.

Amino Acid Sequence↗

PubMed1988

Vaccinia virus late transcripts generated in vitro have a poly(A) head.

A cell free system mediating accurate transcription of vaccinia virus genes was established using lysates of cells in the late phase of infection. Vaccinia late genes are faithfully transcribed in this extract whereas cellular pol II and pol III promoters are not recognized. The late viral transcripts contain a poly(A) head of approximately 35 nt at the 5' end which is not co-linearly encoded in the externally added template. The transcripts obtained in vitro are indistinguishable from the mature in vivo RNAs. The poly(A) head is synthesized de novo and its formation appears to be directly coupled to the transcription of the gene. The synthesis of the poly(A) head transcripts in vitro is consistent with a proposed slippage model.

Base Sequence↗

PubMed1987

Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5' poly(A) leader.

We have demonstrated by primer elongation and cap analysis that mature vaccinia virus late transcripts are discontinuously synthesized. We have shown that RNA transcripts from a translocated 11K and from the authentic 11K and 4b late promoters are extended by approximately 35 nucleotides beyond the "start site" determined by S1 mapping using vaccinia genomic DNA as a probe. Sequencing of the RNA and of the first strand cDNA reveal that a homopolymeric poly(A) sequence is linked to the 5' terminus of the RNA transcripts. S1 mapping of RNA transcripts with a DNA probe containing an A-stretch, replacing promoter sequences upstream of position -1, confirms the existence of a poly(A) leader of approximately 35 A-residues.

Base Sequence↗

PubMed1986

Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation.

We have characterized the vaccinia virus 11-kd late promoter through 5' and 3' deletions and site-directed mutagenesis. The promoter function appears to be contained within an approximately 30-bp fragment, which after translocation is able to direct RNA synthesis late in infection at a reduced level. We demonstrate that a TAAAT sequence in the proximal part of the promoter is essential for its function. This cis-acting element is highly conserved within vaccinia virus late promoters and overlaps the site of transcription initiation. Deletions or mutations within this conserved element completely inactivate the promoter. The evidence indicates that the TAAAT motif functions as a TATA box. The region immediately upstream of the TAAAT motif determines the promoter strength.

Base Sequence↗